[Effect of Berbamine Combined with Ibrutinib on the Proliferation and Apoptosis of Acute Myeloid Leukemia Cells].

OBJECTIVE: To investigate the effects of the combination of berbamine (BBM) and ibrutinib on the proliferation and apoptosis of acute myeloid leukemia (AML) cells and the mechanism of combined action. METHODS: The AML cell lines were treated with BBM, ibrutinib and the combination of the two drugs respectively, CCK-8 method was used to detect the cell proliferation inhibition rate of each group and calculate the combination index (CI). The cell apoptosis in each group was detected by flow cytometry. Western blot was used to determine the expression of related proteins in each group. RESULTS: The cell viability in the combination group was significantly reduced, and the CI value of ED50/ED75/ED90<1. The expression of apoptotic related protein in the combination group was significantly up-regulated, while the expression of p-BTK, p-AKT, CREB, GSK3ß and BCL-XL were significantly down-regulated. CONCLUSION: BBM and ibrutinib can synergistically inhibit the proliferation of AML cells and promote the apoptosis of AML cells. BBM and ibrutinib may play a synergistic effect through the p-BTK/p-AKT/CREB and GSK3ß/BCL-XL signaling pathways.

Analysis of S gene characteristic sequences and changes in properties of protein expression in HBV ASCs with low-level HBsAg.

Objective: We detected the serum HBsAg immune complex (HBsAg-CIC) and sequenced the HBV S gene in these patients to reveal the association between sustained low-level expression of HBsAg and mutated S gene sequence characteristics, protein function changes, and HBsAg immune complex formation. Methods: A total of 204 samples were collected and divided into high-level (n = 60, HBsAg level >10 IU/ml) and low-level (n = 144, HBsAg level ≤ 10 IU/ml) HBsAg groups. The clinical and epidemiological data of the two groups were statistically compared. According to different serological patterns and genotypes, the HBsAg-CIC results of the high-level and low-level HBsAg groups were divided into different subgroups, and then the HBsAg-CIC positive rates among different subgroups were compared. We sequenced the S gene of HBV from the two groups and identified the relevant mutations in the MHR of the S gene. In addition, we compared the changes in HBsAg protein properties and functions after hot spot mutation in the MHR of the S gene. Results: Comparing the positive rates of HBsAg-CIC under different serological patterns and genotypes in the two groups, the HBsAg-CIC positive rate was higher in the low-level HBsAg group. Moreover, there was weak correlation between HBsAg-CIC and HBsAg or HBV DNA in both groups (r = 0.32, 0.27, 0.41, 0.48; P < 0.05). Sequencing of S gene in the two groups, showed that the hot-spot mutations were T126A, M133L/T/S, and F134L/T/I in MHR of S gene of genotype B, and hot-spot mutations were Q101R and I126S/T in MHR of S gene of genotype C. Additionally, the positive rate of MHR mutation in the S gene from HBsAg-CIC positive patients was higher in the low-level HBsAg group. Conclusion: The host immune process of clearing HBV seems to have multiple site mutations in MHR, which changes the physicochemical properties and functions of HBsAg and intensifies the formation of HBsAg-CIC, thus avoiding the effective recognition of HBsAg by the host and resulting in immune tolerance between the host and HBV, which may be one of the formation mechanisms of sustained low-level expression of HBsAg in the serum of HBV-infected persons.

Identification of TLN1 as a prognostic biomarker to effect cell proliferation and differentiation in acute myeloid leukemia.

The protein Talin1 encoded by the TLN1 gene is a focal adhesion-related protein that binds to various cytoskeletal proteins and plays an important role in cell adhesion and movement. Recent studies have shown that it is overexpressed in prostate cancer, liver cancer, and oral squamous cell carcinoma, and is closely related to tumor progression and metastasis. This study integrated bioinformatics and functional analysis to reveal the prognosis and potential functions of TLN1 in AML. The results showed that the expression level of TLN1 was abnormally increased in AML and localized in the cell membrane and cytoplasm, and TLN1 is a significant prognostic indicator of overall survival (OS). Enrichment analysis of related genes showed that TLN1 is related to neutrophil mediated immunity, neutrophil activation and may regulate important signal pathways in hematological tumors including tyrosine kinase receptor, FLT3 and PIK3/AKT. The PPI network shows that TLN1 and MYH9 may be involved in the process of AML tumors together with PIP5K1C, ROCK1, S100A4, MY01A and WAC. Immune infiltration analysis explains that TLN1 is associated with multiple immune cells and may be an important immune marker in AML. Furthermore, molecular biology experiments confirmed that TLN1 is related to the proliferation, differentiation and cycle of AML cells. Silencing TLN1 can inhibit the proliferation of AML cells and promote differentiation through the Talin1/P-AKT/CREB signaling pathway.

[Detection of serum anti-NS2 antibody and recombinant hepatitis C virus nonstructural protein 2 (NS2): its development and evaluation].

Objective To express the recombinant HCV NS2, establish and evaluate the detection method of serum anti-ns2 antibody based on chemiluminescence. Methods Using the NS2 sequence plasmid of HCV 1b genotype (PUC-NS2) as the template, a recombinant plasmid containing the whole NS2 sequence (pGEX-KG-NS2) was constructed. Prokaryotic expression of NS2 protein was induced. The purified NS2 fusion protein was coated on the microplate, and the anti-NS2 antibody detection kit was prepared based on chemiluminescence, and the methodological index was evaluated. Results NS2 fusion protein with relative molecular weight (Mr) of about 51 000 was successfully induced and expressed, and a serum anti-NS2 antibody detection kit was synthesized. Methodological evaluation of kit: Precision test showed favorable results (intra batch coefficient of variation [CV] was 4.60%~9.17%, inter batch CV was 6.62%~10.09%). The relative luminous intensity ratio (RLIR) of the blank limit and the detection limit were 1.57 and 4.80 (r=0.9870), respectively, and the analytical measurement range (AMR) was 1.63~44.50 RLIR. Accuracy experiments: The average recovery was 99.4%. The positive serum samples such as rheumatoid factor had no cross reaction to this test, and the kit was stable within 15 months. The positive rate of anti-NS2 antibody in serum of 45 HCV infected patients was 20% (9/45). Conclusion The prokaryotic expression of HCV NS2 fusion protein is successfully obtained, and the anti-NS2 antibody detection kit in serum is developed.

CaMKIIγ regulates the viability and self-renewal of acute myeloid leukaemia stem-like cells by the Alox5/NF-κB pathway.

Acute myeloid leukaemia (AML) is a frequently fatal malignant disease of haematopoietic stem and progenitor cells. The molecular and phenotypic characteristics of AML are highly heterogeneous. Our previous study concluded that CaMKIIγ was the trigger of chronic myeloid leukaemia progression from the chronic phase to blast crisis, but how CaMKIIγ influences AML stem-like cells remains elusive. In this study, we found that CaMKIIγ was overexpressed in AML patients and AML cell lines, as measured by qRT-PCR and Western blot assays. Moreover, CaMKIIγ decreased when the disease was in remission. Using an shRNA lentivirus expression system, we established CaMKIIγ stable-knockdown AML cell lines and found that knockdown of CaMKIIγ inhibited the viability and self-renewal of AML stem-like cell lines. Additionally, the ratio of CD34 + AML cell lines decreased, and CaMKIIγ knockdown induced the downregulation of Alox5 levels. We further detected downstream molecules of the Alox5/NF-κB pathway and found that c-myc and p-IκBα decreased while total IκBα remained normal. In conclusion, our study describes a new role for CaMKIIγ as a stem-like cell marker that is highly regulated by the Alox5/NF-κB pathway in AML stem-like cells. CaMKIIγ can participate in the viability and self-renewal of AML stem-like cells by regulating the Alox5/NF-κB pathway.

Examination of Ureaplasma urealyticum and Mycoplasma hominis in 4082 Chinese patients

The objective of this study was to analyze the infection rate and drug resistance of Ureaplasma urealyticum (UU) and Mycoplasma hominis (MH) in the genitourinary tract of Chinese patients. From December 2018 to June 2019, vaginal secretion or urinary secretion of outpatients in our hospital were selected for culture and drug sensitivity analysis of Ureaplasma urealyticum and Mycoplasma hominis. In 4082 Chinese samples, 1567 Mycoplasma were detected, a detection rate of 38.39%, among which 1366 cases were UU single positive, accounting for 33.47%, 15 cases were MH single positive, accounting for 0.36%, 186 cases were UU and MH mixed positive, accounting for 4.56%. The most affected age groups were 21-30 years and 31-40 years, accounting for 19.09 and 15.05%, respectively. The results of drug sensitivity showed that doxycycline, minocycline, josamycin, clarithromycin, and roxithromycin were more sensitive to mycoplasma infection. The distribution of Ureaplasma urealyticum and Mycoplasma hominis in the human genitourinary system and their sensitivity to antibiotics is different for sex and age groups.

Examination of Ureaplasma urealyticum and Mycoplasma hominis in 4082 Chinese patients.

The objective of this study was to analyze the infection rate and drug resistance of Ureaplasma urealyticum (UU) and Mycoplasma hominis (MH) in the genitourinary tract of Chinese patients. From December 2018 to June 2019, vaginal secretion or urinary secretion of outpatients in our hospital were selected for culture and drug sensitivity analysis of Ureaplasma urealyticum and Mycoplasma hominis. In 4082 Chinese samples, 1567 Mycoplasma were detected, a detection rate of 38.39%, among which 1366 cases were UU single positive, accounting for 33.47%, 15 cases were MH single positive, accounting for 0.36%, 186 cases were UU and MH mixed positive, accounting for 4.56%. The most affected age groups were 21-30 years and 31-40 years, accounting for 19.09 and 15.05%, respectively. The results of drug sensitivity showed that doxycycline, minocycline, josamycin, clarithromycin, and roxithromycin were more sensitive to mycoplasma infection. The distribution of Ureaplasma urealyticum and Mycoplasma hominis in the human genitourinary system and their sensitivity to antibiotics is different for sex and age groups.

miR-449a promotes liver cancer cell apoptosis by downregulation of Calpain 6 and POU2F1.

Our previous study shows that Calpain 6 (CAPN6) expression is regulated by PI3K-Akt in liver cancer through POU2F1 and CAPN6 which promote cell proliferation and inhibit apoptosis of liver cancer cells. microRNAs (miRNAs) plays important roles in regulation of gene expression. However, whether miRNAs regulates CAPN6 expression and its cellular function is still unknown. This study aims to investigate how miRNAs regulate liver cancer apoptosis through POU2F1-CAPN6. It was verified that POU2F1 could promote cell proliferation and inhibit apoptosis through CAPN6. Using methods of bioinformatics, miR-449a was predicted as a potential regulator of both CAPN6 and POU2F1. It was verified that CAPN6 and POU2F1 were the target genes of miR-449a by luciferase assay. CAPN6 and POU2F1 protein and mRNA levels decreased in liver cancer cells with miR-449a overexpression using western blot and real time RT-PCR assays. miR-449a expression was lower in liver cancer tissues compared with their normal ones, so did the cells. Overexpression of miR-449a inhibited cell proliferation, induced G1 phase arrest and cell apoptosis in liver cancer. Further research demonstrated that miR-449a inhibited cancer cell proliferation and induced apoptosis via suppressing both POU2F1 and CAPN6. The study indicated that miR-449a functions as a tumor inhibitor in liver cancer by decreasing POU2F1 and CAPN6 expression in liver cancer.

Genistein inhibits cell growth and invasion through regulation of miR-27a in pancreatic cancer cells.

BACKGROUND: Although genistein has been reported to exert its anti-tumor activity, the exact mechanism of its action is poorly elucidated. Recently, it has been found that genistein could regulate the expression of microRNAs. Therefore, our aim in the present study was to find whether genistein regulates specific miR-27a in pancreatic cancer cells. METHODS: We performed our studies using multiple methods including MTT assay, RT-PCR, Western blotting analysis, migration, invasion assay, and transfection. RESULTS: We observed that genistein significantly inhibited the expression of miR-27a in pancreatic cancer cells. Moreover, inhibition of miR-27a suppressed cell growth and induced apoptosis as well as inhibited invasion in pancreatic cancer cells. Furthermore, we found a synergy effect between miR-27a and genistein on cell growth inhibition, apoptosis, and invasion, suggesting that targeting miR-27a may represent a potential strategy for treatment of pancreatic cancer. CONCLUSIONS: Our findings demonstrated that genistein plays a tumor suppressor role in part through inhibition of miR-27a in pancreatic cancer cells.

254C>G: a TRPC6 promoter variation associated with enhanced transcription and steroid-resistant nephrotic syndrome in Chinese children.

BACKGROUND: Mutations in canonical transient receptor potential channel 6 (TRPC6) have been identified as responsible for the development of focal segmental glomerulosclerosis, a proteinuric disease with steroid resistance and poor prognosis. This study explores the prevalence of TRPC6 variants in Chinese children with idiopathic nephrotic syndrome (INS), the genotype/phenotype correlation of TRPC6 variants, the therapeutic response, and the underlying molecular mechanism. METHODS: Fifty-one children with sporadic INS were enrolled: 23 steroid-sensitive cases and 28 steroid-resistant cases Polymerase chain reaction was used to amplify 13 exons and the promoter sequences of TRPC6 before sequencing. The expression of TRPC6 in renal tissues was illustrated by immunohistochemistry staining. The transcriptional activity of variants in TRPC6 promoter was measured by the luciferase assay. RESULTS: Three variants (-254C>G, rs3824934; +43C/T, rs3802829; and 240 G>A, rs17096918) were identified. The allele frequency of the -254C>G single-nucleotide polymorphism (SNP) in the steroid-resistant nephrotic syndrome (SRNS) patients (40.5%) was higher than that in the steroid-sensitive nephrotic syndrome subjects (27.1%; P = 0.046). The -254C>G SNP enhanced transcription from TRPC6 promoter in vitro and was associated with increased TRPC6 expression in renal tissues of SRNS patients. CONCLUSION: -254C>G, a SNP underlying enhanced TRPC6 transcription and expression, may be correlated with the development of steroid resistance in Chinese children with INS.

Down-regulation of DNA methyltransferase 3B in staurosporine-induced apoptosis and its mechanism in human hepatocarcinoma cell lines.

Abnormal DNA methylation is one of the important characteristics in tumor cells. Apoptosis plays an essential role in cell survival and processing. It is not clear whether DNA methyltransferases (DNMTs) change in apoptosis and how DNMTs are regulated in apoptosis. In this study, we found that SMMC-7721 or BEL-7404 cells were induced to apoptosis by STS, meanwhile the DNMT3B protein and mRNA level were decreased. To explore the mechanism of DNMT3B down-regulation, we found that the mRNA decay was not changed and core promoter activity of DNMT3B gene was decreased in STS-induced apoptosis. In order to figure out the signal molecule involved in transcriptional regulation of DNMT3B gene by STS, p-JNK, p-ERK, and p-p38 were examined. In STS-induced apoptosis p-JNK level was increased, and p-ERK and p-p38 were decreased. Furthermore, the inhibitor of p-JNK significantly alleviated the decline of DNMT3B protein. We also found that the siRNA of DNMT3B strengthened the cleavage of PARP and pro-caspase-3 as well as up-regulated the p16 gene expression in STS-treated cells. We concluded here that STS-regulated DNMT3B gene expression via p-JNK and down-regulation of DNMT3B-mediated STS-induced apoptosis through the up-regulation p16 expression.

Arsenic trioxide inhibits cell growth and induces apoptosis through inactivation of notch signaling pathway in breast cancer.

Arsenic trioxide has been reported to inhibit cell growth and induce apoptotic cell death in many human cancer cells including breast cancer. However, the precise molecular mechanisms underlying the anti-tumor activity of arsenic trioxide are still largely unknown. In the present study, we assessed the effects of arsenic trioxide on cell viability and apoptosis in breast cancer cells. For mechanistic studies, we used multiple cellular and molecular approaches such as MTT assay, apoptosis ELISA assay, gene transfection, RT-PCR, Western blotting, and invasion assays. For the first time, we found a significant reduction in cell viability in arsenic trioxide-treated cells in a dose-dependent manner, which was consistent with induction of apoptosis and also associated with down-regulation of Notch-1 and its target genes. Taken together, our findings provide evidence showing that the down-regulation of Notch-1 by arsenic trioxide could be an effective approach, to cause down-regulation of Bcl-2, and NF-κB, resulting in the inhibition of cell growth and invasion as well as induction of apoptosis. These results suggest that the anti-tumor activity of arsenic trioxide is in part mediated through a novel mechanism involving inactivation of Notch-1 and its target genes. We also suggest that arsenic trioxide could be further developed as a potential therapeutic agent for the treatment of breast cancer.

Sp1 is involved in regulation of cystathionine γ-lyase gene expression and biological function by PI3K/Akt pathway in human hepatocellular carcinoma cell lines.

Hydrogen sulfide (H(2)S) has been found to play an important role as a novel gasotransmitter involved in many biological processes. The regulatory role of endogenous H(2)S-producing enzyme on cancer cell survival is complex and unclear. According to the data that cystathionine γ-lyase (CSE) gene, catalyzed H(2)S production in trans-sulfuration pathway, was upregulated in Akt stably transformed mouse embryonic fibroblast cells, the mechanisms that elevated CSE expression by PI3K/Akt signaling pathway and its biological functions in cell survival were studied. In the present study, firstly, the results showed that PI3K/Akt positively correlated with CSE expression levels in human hepatocellular carcinoma cell lines. CSE expression was decreased by the PI3K inhibitor or Akt deletion, while upregulated with the activating of Akt. Based on dual-luciferase reporter assay, the -592/+139 gene fragment represented the CSE core promoter, and the PI3K/Akt pathway regulated CSE expression on transcriptional level. Sp1 was the critical transcription factor in regulation of CSE expression via the mutation of transcription factor binding sites on the promoter. Furthermore, we proved that Sp1 could directly bind to CSE promoter by ChIP assay. In addition, we explored that the endogenous H(2)S production was connected with the regulated CSE expression, and CSE/H(2)S promoted human hepatocellular carcinoma cell proliferation via cell cycle progression regulation. In summary, we have, for the first time, demonstrated that PI3K/Akt pathway regulates the CSE expression via Sp1, which is particularly important to understand the effect of PI3K/Akt and CSE on the tumorigenesis.

The PI3K-Akt pathway regulates calpain 6 expression, proliferation, and apoptosis.

The calpains are a family of cysteine proteases involved in some biological processes whose activities are highly dependent on Ca(2+). Calpain 6 (CAPN6), one member of the family, is unique in that it lacks the active-site cysteine residues for protease activity. According to the data that CAPN6 was up-regulated in the Akt transformed mouse embryonic fibroblast cells by cDNA chip, the mechanisms underlying elevated CAPN6 expression by PI3K-Akt signaling pathway and its biological functions were studied. The results showed that CAPN6 was down-regulated on transcriptional and post-transcriptional levels by the PI3K inhibitor or Akt deletion. CAPN6 protein was stabilized by PI3K-GSK-3ß pathway. Deleted CAPN6 promoters activity were assessed by dual-luciferase reporter system, and the founding indicated that -93/+200 DNA fragment was the core promoter of it. Transcription factor binding sites in the CAPN6 promoter were mutated and the results showed that AP1, Oct-1, and FoxD3 were the critical transcription factors in regulation of CAPN6 expression. In addition, CAPN6 promoted cancer cell proliferation and inhibited its apoptosis. The finding demonstrates that CAPN6 is regulated by the PI3K-Akt signaling pathway and provides evidence that it may be a therapeutic target of cancer.

The effect of epidermal growth factor receptor variant III on glioma cell migration by stimulating ERK phosphorylation through the focal adhesion kinase signaling pathway.

Epidermal growth factor receptor variant III (EGFRvIII), the most common EGFR mutation, is associated with cell migration of glioblastoma multiforme (GBM) cases; however, the mechanism has not been elucidated. In this study, we found that the EGFRvIII-promoted glioma cell migration was closely linked to high levels of tyrosine phosphorylation in focal adhesion kinase (FAK) Y397. We also demonstrated that EGFRvIII formed a complex with FAK, resulting in enhanced tyrosine phosphorylation levels of FAK Y397 and EGFR Y1068. After knockdown of FAK expression via anti-FAK shRNA, the U87ΔEGFR cell migration was significantly inhibited, accompanying with the reduced phosphorylation levels of extracellular signal-regulated kinase (ERK1/2). Furthermore, the role of ERK1/2 in FAK-regulated cell migration was confirmed. Taken together, our results suggest that FAK and its downstream molecule ERK were involved in EGFRvIII-promoted glioma cell migration in U87ΔEGFR cells.

Transcriptional and post-transcriptional control of DNA methyltransferase 3B is regulated by phosphatidylinositol 3 kinase/Akt pathway in human hepatocellular carcinoma cell lines.

DNA methyltransferases (DNMTs) are essential for maintenance of aberrant methylation in cancer cells and play important roles in the development of cancers. Unregulated activation of PI3K/Akt pathway is a prominent feature of many human cancers including human hepatocellular carcinoma (HCC). In present study, we found that DNMT3B mRNA and protein levels were decreased in a dose- and time-dependent manner in HCC cell lines with LY294002 treatment. However, we detected that LY294002 treatment did not induce increase of the degradation of DNMT3B protein using protein decay assay. Moreover we found that Akt induced alteration of the expression of DNMT3B in cells transfected with myristylated variants of Akt2 or cells transfected with small interfering RNA respectively. Based on DNMT3B promoter dual-luciferase reporter assay, we found PI3K pathway regulates DNMT3B expression at transcriptional level. And DNMT3B mRNA decay analysis suggested that down-regulation of DNMT3B by LY294002 is also post-transcriptional control. Furthermore, we demonstrated that LY294002 down-regulated HuR expression in a time-dependent manner in BEL-7404. In summary, we have, for the first time, demonstrate that PI3K/Akt pathway regulates the expression of DNMT3B at transcriptional and post-transcriptional levels, which is particularly important to understand the effects of PI3K/Akt and DNMT3B on hepatocarcinogenesis.

EGF-mediated migration signaling activated by N-acetylglucosaminyltransferase-V via receptor protein tyrosine phosphatase kappa.

N-acetylglucosaminyltransferase-V (GnT-V) has been reported to be closely associated with tumor migration, but the mechanism has been not currently clear. In this study we found that GnT-V activated EGFR signaling and promoted cell migration through receptor protein tyrosine phosphatase kappa (RPTPkappa). The overexpression of GnT-V gene in human hepatoma SMMC-7721 cell line not only induced the addition of beta1,6 GlcNAc branch to N-glycan of RPTPkappa but also decreased the protein level of RPTPkappa, which both contributed to the decreased phosphatase activity of RPTPkappa activating subsequently EGFR signaling. Moreover, we found that the knockdown of RPTPkappa and its addition of beta1,6 GlcNAc branch both promoted cell migration, which could be ascribed to the activation of EGFR signaling regulated by RPTPkappa. Therefore, our findings could provide an insight into the molecular mechanism of how GnT-V promoted cell migration, suggesting that RPTPkappa could be one of factors regulating the EGF-mediated migration signals.